This proposal has the following goals. The chirality of presqualene pyrophosphate will be determined by synthesis of an optically active presqualene alcohol. A number of substrate analogues will be synthesized, labeled with tritium or C14, and incubated with the squalene synthetase. These analogues have been selected since each would not be predicted to be metabolized by all the proposed mechanisms. However, each can be metabolized by at least one. Comparison of the products should indicate which of the proposed pathways, if any, account for squalene biogenesis. The "real" role of presqualene pyrophosphate, intermediate or alternate precursor, will be established. Artificial substrates will be employed to trap the proposed covalently bound enzyme substrate intermediate. This result will permit the identification of the catalytic nucleophile at the active site. The existence of other intermediates involved in the conversion of presqualene pyrophosphate to squalene will also be determined through study of analogues. From this knowledge, further regulation of cholesterol biogenesis may result by utilizing drugs specific for the squalene synthetase.